Project Summary and Abstract: Type 1 Diabetes (T1D) affects more than 1 million people in the United States and is usually diagnosed in children and can lead to blindness, heart disease and kidney failure. A major focus of intervention for T1D is on the detection and characterization of autoreactive T cells, which play a central role in the attack on insulin producing islet cells. For medical intervention for T1D to be most effective, the disease should be detected and treated before the onset of symptoms. Current methods to identify the targets of autoreactive T1D-specific T cells are slow, technically demanding, labor- and reagent-intensive, and consume large numbers of T cells to test limited numbers of targets. Sample size is an extremely important consideration in T1D and is often a limiting factor when testing clinical samples, especially as young children and adolescents are unable to give large samples. The objective of this SBIR proposal is to develop an automated T cell analysis assay to identify and functionally characterize autoreactive T1D antigen-specific CD4+ and CD8+ T cells capable of testing thousands of targets simultaneously using as little as 2ml of patient sample. The proposed system is based upon integrating patent pending MHC-peptide array technology with an automated flow cell detection and analysis system. The innovative approach of this project will combine MHC-peptide arrays in a self-contained sample cartridge with a highly parallel luminescent detection system that is capable of rapid, multiplexed analysis of T1D antigen-specific T cells using dramatically smaller sample volumes than current methods. The major advancements of this system will be the ability to visually detect antigen-specific CD4+ and CD8+ autoreactive T cells binding to MHC-peptide-complexes and to correlate MHC: T cell receptor binding to resulting activation and secretion of multiple cytokine effector molecules. The goal is to create a T cell analysis system that offers high throughput screening, multi-parametric characterization capability and incorporates state-of- the-art integrated sample handling for ease-of-use. At present, there are no systems available that offer the breadth of capabilities, the simplicity of use and limited sample size requirements as the proposed T cell analysis system using MHC-peptide arrays in the fight against T1D.